Mass spectrometry (MS) is one analytical chemistry technique that helps to identify the amount and type of chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions. There are several steps required to perform MS analysis on complex samples. In some uses of MS, these steps include extraction, pre-concentration, and sample ionization. These steps can cause certain MS tests to be time-consuming, expensive, and complex.
MS is the gold standard for performing trace analysis in complex mixtures, but many laborious sample preparation steps generally must be carried out in the laboratory to perform the analyses.
Ion generation using wetted porous material has simplified certain applications of MS. Exemplary systems and methods for ion generation using wetted porous material, such as, for example, paper-spray ionization, have been disclosed and claimed in U.S. Pat. No. 8,859,958, issued Oct. 14, 2014, the entire disclosure of which is hereby expressly incorporated herein by reference.
Paper-spray MS could have a significant impact as a rapid and simple method to assess patient compliance in methadone clinics, pain management clinics, in psychiatric patients, etc. While immunoassays are widely used for this purpose, their limitations for drug screening are well-known, including inadequate sensitivity and selectivity, which can result in false negatives and unacceptably high false positive rates. Faster and simpler methods for analyzing organic contaminants such as pharmaceuticals, abused drugs, pesticides, and personal care products in environmental samples are needed. River or ground water monitoring is challenging because the detection limits required are low, often in the low part per trillion (ppt) range. Improvements in selectivity and on-cartridge sample preparation methods would be a significant improvement over existing methods.
Driven by the desire to increase sample throughput and to perform MS based assays outside of a traditional analytical laboratory, there is a strong and growing interest in developing MS methods that are faster, simpler, and require less or even no sample preparation prior to analysis. Progress in this field accelerated rapidly with the discovery of two “ambient ionization” or “direct analysis” methods in 2004 and 2005, so-called because samples can be analyzed directly in ambient conditions without sample preparation. The two methods, desorption electrospray ionization (DESI) and direct analysis in real time (DART), helped to spark a new research field within the analytical community that has resulted in an array of new applications and new technologies that dramatically simplify MS based assays.
Paper-spray MS is a method for performing rapid, direct analysis of samples spotted on paper or another porous substrate. A liquid sample (e.g., blood or other biofluids, waste water, etc.) is spotted onto the paper and stored as a dried spot. Analysis is performed by depositing a small volume of solvent to the paper where it wicks through the porous substrate and sample by capillarity. The paper, which in some instances is cut to a sharp point, is positioned a few millimeters away from the atmospheric pressure inlet of a mass spectrometer and a high voltage (3-5 kV) is then applied directly to the paper, inducing an electrospray at the tip of the paper. The solvent evaporates from the charged droplets generated by the electrospray process, leaving gas phase ions of the analyte molecules which can then be detected by a mass spectrometer. Chemicals which are both soluble in the extraction/spray solvent and ionizable will be detected immediately by the mass spectrometer provided they are sufficiently concentrated.
Analytes are detected immediately if they are not appreciably retained by the short distance of substrate they travel through. The entire analysis takes about 60 seconds and requires only the paper substrate on which the sample is already stored, a small amount of solvent, and an electrical connection to a low-current, high voltage power supply.
Paper-spray has a number of advantages for performing rapid chemical analysis of complex samples by MS. Paper-spray requires no sample preparation. Direct analysis of blood, urine, and waste-water, has been demonstrated. Single digit ng/mL or sub-ng/mL detection limits for drugs, pharmaceuticals and other small molecules from these complex matrices are currently routine on commercial triple quadrupole mass spectrometers. The sample volume required for paper-spray is low. Methods published in the literature use between 0.5 μL and 15 μL of sample. The small sample consumption of paper-spray is a significant advantage for volume limited samples.
Moreover, the paper substrate doubles as an ionization source and as a sample storage medium. Storage of dried biofluid samples in particular is known to improve sample stability at room temperature. The paper substrate is inexpensive and readily available. Clogging, which commonly occurs in conventional capillary electrospray ionization, is unlikely in paper-spray due to the multi-porous nature of the substrate. Carryover is not a problem because the ion source and everything that contacts the sample is discarded after each analysis. The amount of solvent required per sample is low (less than 100 μL), and all of the solvent is consumed so there is no solvent waste to dispose.
Also, the need for liquid chromatography is removed, which simplifies the analysis and removes common sources of failure in HPLC-MS assays, such as leaks and clogged columns.
Much of the early work on paper-spray MS has focused on the targeted quantitative analysis of drugs and drug metabolites directly from dried biofluids. In this application, an isotopically labeled internal standard (IS) is typically mixed with the blood prior to deposition onto the paper. Signals for the analytes and the corresponding IS's are then determined. Quantitation is achieved by simultaneously monitoring the analyte and the IS as they are extracted and ionized directly from the paper substrate. Due to the complexity of the matrix and the low drug levels, tandem MS (MS/MS) or high-resolution MS (HRMS) was done for this application. Multiple analytes can be simultaneously quantitated as long as analytes have different masses or generate different fragment ions during MS/MS. A summary of some of the paper-spray methods developed for the quantitative analysis of drugs from dried biofluid samples by paper-spray MS/MS are shown in Table 1 below.
TABLE 1Some paper-spray methods for the quantitativeanalysis of drugs from dried blood.AssayApproximateMolecularrangeLODRefer-DrugIonIS(ng/mL)(ng/mL)enceImatinib[M + H]+[H]8- 4-80000.7(1)imatinibMelphalan[M + H]+[H]8-100-2500020(2)melphalanPazopanib[M + H]+[H]4-100-500003(3)pazopanibTacrolimus[M + Na]+[H]3-1.5-30  0.08(4)tacrolimusCocaine[M + H]+[H]3-10-800 0.05(5)cocainenicotine[M + H]+[H]3-1-1000.3(6)nicotine
A number of other applications have been reported for paper-spray MS as well. These include profiling of lipids in bacteria and microalgae, online chemical monitoring of cell culture, detection of chemical contaminants in food, including plasticizers, melamine, pharmaceuticals, and 4-methylimidazole, analysis of acyl-carnitines from blood and urine, and as an ion source for a microfluidic chip.
Analyte chemical and physical properties and the type of sample matrix both significantly affect the limits of detection for some paper-spray MS assays. In one comparative analysis of numerous small molecules, with a molecular weight range of 150 to 850, in blood samples with widely varying properties, the limit of detection (LOD) varied over four orders of magnitude. The chemical matrix also significantly affects the LOD, with poorer signal intensity seen in dirtier matrices such as urine, waste water, and plasma. In a typical paper-spray MS analysis, increasing the sample volume beyond a couple of microliters does not improve detection limits because the size of the paper substrate and the volume of extraction/spray solvent has to be increased as well. Concentration of the analyte and/or removing some of the matrix components that cause ion suppression is needed to improve detection limits.
Paper-spray has the potential to dramatically simplify and expand the utility of mass spectrometric assays. There are a number of limitations that need to be addressed if the field is to move forward, however. First, the detection limits are often inadequate. While low or sub-ng/mL detection limits can be achieved in favorable cases, detection limits are significantly higher for chemical analytes that do not ionize as efficiently or cannot be recovered from the sample matrix as well. Additionally, detection limits are significantly higher on portable or miniature mass spectrometers due to size constraints limiting the MS performance. Performing paper-spray on challenging analytes, on applications where lower detection limits are required, or on portable mass spectrometers will require sample preparation methods to pre-concentrate the analytes or remove interfering matrix components.
Second, there is a lack of simple approaches for incorporating an IS into the sample, which is required for quantitative analysis and quality control. All of the assays in Table 1 mixed an IS solution into the liquid sample prior to spotting the sample onto the paper substrate. This raises a number of problems. If this operation is done at the point of collection, accuracy cannot be assured. If, on the other hand, the liquid sample is shipped to a lab so that a technician can perform this operation, then a significant advantage of paper-spray MS has been lost (the ease and stability of transporting samples as dried sample spots).
Third, in the case of drug, pharmaceutical, or metabolite measurement plasma is often preferred over blood. Plasma isolation is generally carried out by drawing venous blood into a collection tube by a phlebotomist and centrifuging the blood collection tube. In addition to being labor intensive, this approach requires that the blood be stored and transported as a liquid for several hours, which can be a problem for some unstable analytes. In addition, collection of blood and isolation of plasma in this manner is not possible in resource limited settings.
Another limitation of direct MS analysis methods is inadequate selectivity. Because there is no chromatography prior to the MS analysis, chemical discrimination in paper-spray and other direct MS analysis methods must occur by MS/MS or by HRMS alone. Isomers cannot be distinguished by HRMS. In some cases, MS/MS can distinguish structural isomers and even quantitate them simultaneously as long as they fragment differently upon collisional activation and have unique fragment ions. Closely related structural isomers, however, frequently fragment so similarly that no unique fragment ions exist.
Thus, there is still a need for simple, fast, and low-cost devices and methods for carrying out MS by ion generation using wetted porous materials, such as by paper-spray ionization.